Alzheimer's disease (AD) is a neurodegenerative disorder of the brain, which is accompanied at the cellular level by a massive loss of neurons in the limbic system and in the cerebral cortex. In the brain areas affected, protein deposits, so-called plaques, can be detected at the molecular level, which are an essential characteristic of Alzheimer's disease. The protein occurring most frequently in these plaques is a peptide of 40 to 42 amino acids, which is designated as Aβ-peptide. This Aβ-peptide is a cleavage product of a significantly larger protein of 695 to 770 amino acids, the so-called amyloid precursor protein (APP).
APP is an integral transmembrane protein, which firstly traverses the lipid bilayer. By far the largest part of the protein is extracellular, while the shorter C-terminal domain is directed into the cytosol (FIG. 1). The Aβ-peptide is shown dark-gray in FIG. 1. About two thirds of the Aβ-peptide originates from the extracellular domain and about one third from the transmembrane domain of APP.
Beside the membrane-based APP, a secreted form of the amyloid precursor protein can be detected which consists of the large ectodomain of the APP and is designated as APPsec (“secreted APP”). APPsec is formed from APP by proteolytic cleavage, which is effected by the α-secretase. The proteolytic cleavage takes place in a site of the amino acid sequence of APP, which is within the amino acid sequence of the Aβ-peptide (after amino acid residue 16 of the Aβ-peptide). Proteolysis of APP by the α-secretase thus excludes the formation of the Aβ-peptide.
The Aβ-peptide can thus only be formed from APP in an alternative processing route. It is postulated that two further proteases are involved in this processing route, one protease, which is designated as β-secretase, cleaving at the N-terminus of the Aβ-peptide in the APP and the second protease, which is designated as γ-secretase, releasing the C-terminus of the Aβ-peptide (Kang, J. et al., Nature, 325, 733) (FIG. 1).
To learn more about the secretases (α-secretase, β-secretase, γ-secretase) is of great interest, in particular in the context of investigations on Alzheimer's disease, e.g., for the identification of the secretases or factors involved in secretase regulation and Aβ-peptide formation (Wolfe, M. S. (2001), J. Med. Chem., 44(13), 2039-2060). The inhibition of β-secretase and in particular of γ-secretase could lead to a reduction in the Aβ-production, on the other hand an activation of the α-secretase could increase the processing of APP in APPsec and would thus simultaneously reduce the formation of the Aβ-peptide. A transgenic C. elegans, which is found in the course of such investigations is described in the U.S. Pat. No. 6,673,600, the contents of which are incorporated herein by reference.
There are many indications that the Aβ-peptide (Aβ) is a crucial factor in the occurrence of Alzheimer's disease. Inter alia, neurotoxicity of Aβ-fibrils in cell culture is postulated (Yankner, B. A. et al., (1990) Proc Natl Acad Sci USA, 87, 9020). In patients with Down's syndrome, in which the gene encoding APP occurs in an additional copy, the neuropathology characteristic of Alzheimer's disease also occurs even at an age of 30 years. Here, it is assumed that the overexpression of APP follows an increased conversion into the Aβ-peptide (Rumble, B. et al., (1989), N. Engl. J. Med., 320, 1446). great
Probably the strongest indication of the central role of the Aβ-peptide is the familial forms of Alzheimer's disease. Here, mutations are found in the APP gene around the area of the β- and γ-secretase cleavage sites or in two further AD-associated genes (presenilins), which in cell culture lead to a significant increase in Aβ-peptide production (Scheuner, D. et al., (1996), Nature Medicine, 2, 864).
There are a number of indications of the fact that APP is firstly cleaved into the Aβ-peptide by the β-secretase during its processing in order to serve subsequently as a substrate for γ-secretase. The γ-secretase therefore has a crucial role in the formation of the Aβ-peptide (Wolfe, M. S. (2001), loc.cit).
In general, the detection of Aβ-peptide is difficult, since only a small amount of APP is converted (Simons M, et al., Neurosci (1996) 1; 16(3):899-908). Moreover, the Aβ-peptide is a very small fragment of about 4 kDa, which has a great tendency to self-aggregation due to its hydrophobic character. Accordingly, Aβ-peptide easily precipitates under physiological conditions (Hilbich, C. et al., (1991) J. Mol. Biol., 218, 149) and is in its precipitated form not available for detection.
The detection of the Aβ-peptide in eukaryotic cells is carried out by means of immunobiological methods such as, e.g., ELISA, immunoprecipitation and Western blotting (Suzuki, N. et al., Science 1994, 27, 264(5163) 1336; Haass, C. et al., (1992) Nature, 359, 322). Further, an in vitro assay for the determination of γ-secretase activity from purified membrane fractions containing PS1 (presenilin 1) was described by Wolfe et al. (1999). These processes are very time consuming, as they involve incubation steps with appropriate antibodies, steps destroying the cells obtained from suitable cell culture or model organisms (e.g., C. elegans). The said methods are not suitable in an automated assay system, e.g., for high throughput screening, to identify compounds, which specifically inhibit or decrease the activity of a γ-secretase. In part, this is because γ-secretase activity is dependent upon an assembly of proteins (Mattson, (2003) Nature 422, 385), which is, to date, only active in a complex membrane lipid environment.
Further, the activity of the γ-secretase can be demonstrated according to the teachings of WO00/34511A2, the contents of which are incorporated herein by reference, which describes a process for the determination of γ-secretase activity and for the detection of γ-secretase by the detection of the Aβ-peptide. The process of WO00/34511A2 utilizes a transgene which encodes a fusion protein comprising: the amino acid sequence GAIIGLMVGGVVIATVIVITLVML (SEQ ID NO. 1) as the enzymatic target site of γ-secretase, a signal peptide (SP) at the 5′-end, a promoter and, if appropriate, further coding and/or non-coding nucleotide sequences, which is incorporated into a cell in order to express the said fusion protein.
When the fusion protein is specifically cleaved by the γ-secretase present in the cell, a first partial protein is formed, containing the amino acid sequence GAIIGLMVGGVV (SEQ ID NO. 2), and a second partial protein is formed, containing the amino acid sequence VIVITLVML (SEQ ID NO. 3). Subsequently, the said first and/or second partial protein is detected, e.g., by use of a suitable reporter, which is, e.g., a reporter gene, which is activated by the release of a transcription activator coupled to the first and/or second partial protein.
Due to the known problems accompanied with the detection of Aβ-peptide, it is the problem of instant invention to improve the process of WO00/34511A2, e.g., by decreasing the background signal and/or increasing the signal specificity, in order to improve the signal/noise ratio in the assay of the invention.